The retina in lower vertebrates shows a remarkable regenerative ability that is lost in mammals. Multipotent progenitor cells, that are capable of differentiating into a variety of retinal cell types, have been isolated from the ciliary margin and retina of mammalian eyes. Despite their presence, these progenitor cells are normally quiescent and unable to regenerate damaged retina. This proposal will investigate the ability of microvesicles, released from mouse embryonic stem cells, to reactivate mouse retinal progenitor cells. The long term goal of this project is to discover novel ways of reactivating quiescent progenitor cell populations in the human eye, so that regeneration of damaged retina may be possible. Microvesicles are plasma-membrane particles that are released into the extracellular environment. Very recently, microvesicles have been reported from embryonic stem cells cultured in vitro. Our preliminary results show that these embryonic stem cell microvesicles contain RNA and protein. Most interestingly, they contain a specific class of RNA molecules called microRNAs, which are potent regulators of translation. Microvesicles may serve a role in intercellular communication in one of several manners. They may transfer microRNAs, mRNAs, or proteins to cells. Alternatively they can signal cells through surface proteins found on microvesicles. Our first aim is to characterize the RNA and protein contents of mouse embryonic stem cell microvesicles to look for candidates that might alter stem cell programming. We will use microarray analysis and qRT-PCR for mRNA and microRNA profiling, and mass spectrometry, Western blot analysis, and immunocytochemistry for protein profiling. We will also explore in Aim I the ability of these microvesicles to directly transfer RNA or protein to cells in vitro. Our second aim is to determine if these microvesicles can activate the quiescent stem cell population found in the ciliary margin and retina of mouse eyes. We will inject microvesicles into the aqueous, vitreous, and subretinal space of mice and look for increased proliferation of stem cells with BrDU- labeling. Our final aim is to look for endogenous microvesicles in the aqueous and vitreous and also to characterize them using microarray profiling, qRT-PCR, mass spectrometry, Western blot analysis, immunocytochemistry, and electron microscopy. The information generated in this proposal will not only lead to an increased understanding of the role that extrinsic mRNA, microRNAs, and proteins play in determining stem cell fate, but may also identify microvesicles as novel endogenous signaling factors in the eye, and possibly contributing to the stem cell niche.